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Molecular cloning and biochemical characterization of a myotoxin inhibitor from Bothrops alternatus snake plasma |
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| Authors: | Santos-Filho Norival A Fernandes Carlos A H Menaldo Danilo L Magro Angelo J Fortes-Dias Consuelo L Estevão-Costa Maria Inácia Fontes Marcos R M Santos Camila R Murakami Mário T Soares Andreimar M |
| Institution: | a Departamento de Bioquímica e Imunologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, FMRP-USP, Ribeirão Preto-SP, Brazil b Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, FCFRP-USP, Ribeirão Preto-SP, Brazil c Departamento de Física e Biofísica, Instituto de Biociências, Universidade Estadual Paulista, UNESP, Botucatu-SP, Brazil d Diretoria de Pesquisa e Desenvolvimento, Fundação Ezequiel Dias, Belo Horizonte-MG, Brazil e Laboratório Nacional de Biociências, Centro Nacional de Pesquisas em Energia e Materiais, Campinas, São Paulo, Brazil |
| Abstract: | Phospholipases A2 (PLA2s) are important components of Bothrops snake venoms, that can induce several effects on envenomations such as myotoxicity, inhibition or induction of platelet aggregation and edema. It is known that venomous and non-venomous snakes present PLA2 inhibitory proteins (PLIs) in their blood plasma. An inhibitory protein that neutralizes the enzymatic and toxic activities of several PLA2s from Bothrops venoms was isolated from Bothrops alternatus snake plasma by affinity chromatography using the immobilized myotoxin BthTX-I on CNBr-activated Sepharose. Biochemical characterization of this inhibitory protein, denominated αBaltMIP, showed it to be a glycoprotein with Mr of ∼24,000 for the monomeric subunit. CD spectra of the PLA2/inhibitor complexes are considerably different from those corresponding to the individual proteins and data deconvolution suggests that the complexes had a relative gain of helical structure elements in comparison to the individual protomers, which may indicate a more compact structure upon complexation. Theoretical and experimental structural studies performed in order to obtain insights into the structural features of αBaltMIP indicated that this molecule may potentially trimerize in solution, thus strengthening the hypothesis previously raised by other authors about snake PLIs oligomerization. |
| Keywords: | Phospholipase A2 Myotoxin inhibitor cDNA Protein modeling Molecular dynamics Bothrops alternatus Snake plasma |
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